The technique of nucleic acid hybridization has been successfully employed for the study of DNA structure, nucleic acid purification, gene localization, and detection and diagnosis of diseases and mutations.
Hybridization assays are based on the structural properties of DNA molecules. The DNA of most organisms is comprised of two strands of polynucleotides which associate largely through noncovalent interactions into the familiar double helical structure. The most important of these noncovalent interactions is hydrogen bonding between adenine and thymine and between cytosine and guanine. It was
demonstrated by Britten, et al. (Sci. American 222(4):24-31 (1968)) that under certain conditions it was possible to cause the two strands to separate from one another. This process of strand separation has been variously referred to as unwinding, denaturing or melting of the double-stranded duplex. It was further discovered that under a second set of conditions the strands would reassociate to reform the duplex DNA structure, this process being referred to as annealing, reassociation or renaturation.
It is also now known to be possible to denature DNAs from two different sources, then mix the two populations of single stranded nucleic acids, and under renaturation conditions estimate the percentage of double stranded hybrids formed, thus providing an indication of sequence homology between the two sources. The double-stranded molecules formed are hybrids, and the process is known as DNA hybridization. In a similar manner, a small nucleotide segment ranging from a fraction of a single gene up to a size which would include several genes may be used to hybridize to DNA for the purposes of determining whether a complementary segment is present in the sample, and if so, where it is located. The segment of interest is often of a predetermined sequence or function and is generally referred to as a nucleic acid hybridization probe.
Such probes have become extremely important as reagents for the detection of specific nucleic acid sequences. Commonly the probes are labelled with radioactive isotopes to facilitate their analytical detection. The isotopes normally employed include .sup.32 P, .sup.125 I or .sup.3 H; however, considerations regarding stability, safety, ease of detection and disposal of waste have fostered the development of non-isotopically labelled probe molecules.
One approach has been to detect nucleic acids by immunological means, either by developing antibodies which will discriminate between single and double stranded DNAs or by labelling the nucleic acid with an immunoreactive component such as a hapten. Landegent, et al. (Exp. Cell Res. 153:61-72 (1984)) and Tchen et al. (Proc. Nat'l Acad. Sci. USA 81:3466-3470 (1984)) have employed N-acetoxy-N-2-acetylaminofluorene to label probes. These probes can be detected by direct or indirect enzyme-linked immunosorbent assays (ELISA). Because of the carcinogenic nature and attendant disposal problems associated with N-acetoxy-N-2-acetylaminofluorene, alternative methods are desired.
A hapten which has gained widespread use for labeling nucleic acid molecules is the vitamin, biotin. Of particular advantage is the high affinity (k.sub.d =.sup.-15 M) between biotin and the glycoprotein avidin (Green, N. M., Adv. Protein Chem. 29:85-133 (1975)). This affinity is stronger than that between a typical antibody and a typical antigen. Moreover, it was found that avidin could be reacted with enzymes, fluorescent groups or electron dense molecules to form analytically detectable avidin-conjugates.
Ward, et al. (Proc. Nat'l Acad. Sci. USA 79:4381-4385 (1982) and Proc. Nat'l Acad Sci. USA 80:4045-4049 (1983)) have developed a method for labeling nucleic acids with biotin. Biotin-labelled analogs of nucleic acid precursors such as dUTP and UTP were enzymatically incorporated into nucleic acids. The method of Ward et al. requires expensive substrates and enzyme, and large scale preparation of biotin labelled nucleic acids by this method is economically disadvantageous. It was desirable, therefore, to develop chemical methods for labelling nucleic acid with biotin.
Several attempts to develop chemical labelling methods have been reported. Manning et al. (Chromosoma 53:107-117 (1975)) have disclosed the chemical cross-linking of a biotin labelled cytochrome C conjugate to RNA with formaldehyde. M. Renz and C. Kurz substituted enzymes such as peroxidase or alkaline phosphatase for cytochrome C in a similar crosslinking procedure (Nucleic Acid Res. 12(8):3435-3444 (1984)). However, the conjugates tend to be unstable under hybridization conditions. Moreover, the adducts to the nucleic acid often present serious steric hindrance to the hybridization process.
Finally, Forster, et al. (Nucleic Acid Res 13(3):745-761 (1985)) have disclosed the synthesis of a photoactivatable biotin analog of the formula: ##STR1## which may be used to label M13 DNA probes. However, this compound reacts with both single and double stranded DNA and as pointed out by the authors, this dual reactivity limits the extent of probe modification possible without interfering with the hybridization of target sequences by single stranded regions of the probe.
Carbodiimides have been previously reported to be useful for peptide synthesis (J. C. Sheehan et al., J. Org. Chem. 21:439 (1956)). The reactivity of carbodiimides with guanine and thymidine nucleotides has been reported by P. T. Gilham (J. Am. Chem. Soc. 84:688 (1962)).